Technical TECHNICALreports: W REPORTSaste Management Pathogens and Indicators in United States Class B Biosolids: National and Historic Distributions Ian L. Pepper* University of Arizona John P. Brooks USDA–ARS Ryan G. Sinclair Loma Linda University Patrick L. Gurian Drexel University Charles P. Gerba University of Arizona ewage sludge is defined as “the solid, semisolid, or liquid resi- This paper reports on a major study of the incidence of due generated during the treatment of domestic sewage in a indicator organisms and pathogens found within Class B S biosolids within 21 samplings from 18 wastewater treatment treatment works.” In contrast, biosolids, as defined by a National plants across the United States. This is the first major study Research Council Committee (2002) that addressed the health of its kind since the promulgation of the USEPA Part 503 effects of biosolids, is the term given to the end product that Rule in 1993, and includes samples before and after the Part results from treatment of sewage sludge to meet the land-appli- 503 Rule was promulgated. National distributions collected cation standards of the USEPA Part 503 Rule (USEPA, 1993). between 2005 and 2008 show that the incidence of bacterial and viral pathogens in Class B mesophilic, anaerobically Depending on the level of treatment, two classes of biosol- digested biosolids were generally low with the exception of ids are produced: Class A biosolids (higher level of treatment- adenoviruses, which were more prevalent than enteric viruses. processes designed to further reduce pathogens), which contain No Ascaris ova were detected in any sample. In contrast, no detectable levels of pathogens in 4 g of dry solids; or Class B indicator organism numbers were uniformly high, regardless of biosolids (lower level of treatment-processes designed to reduce whether they were bacteria (fecal coliforms) or viruses (phage). Indicators were not correlated with pathogen loads. Historic pathogens), which routinely contain bacterial, parasitic, and viral distributions were collected between 1988 and 2006 at one pathogens (Table 1). Approximately 5.5 billion kg (6 million dry location in Tucson, AZ. By comparing data collected before tons) of biosolids are produced annually in the United States, of and after 1993, the influence of the USEPA Part 503 Rule which 60% are used for land application, with the vast majority on indicator and pathogen levels within Class B biosolids can of it being Class B biosolids (NRC, 2002). be inferred. In general, the bacterial indicators total and fecal coliforms decreased from the 1980s to present. Enteric virus The greatest amount of uncertainty in quantitative microbial concentrations after 1993 are much lower than those reported risk assessment is due to the lack of data on exposure to patho- in other studies in the 1980s, although our values from 1988 gens (Haas, 1996), and to properly assess the risk from land-applied to 1993 are not significantly different from our values obtained Class B biosolids, it is important to know the number of pathogens from 1994 to 2006. Presumably this is due to better and more in the biosolids after treatment and before land application. A pre- consistent treatment of the wastewater, illustrating that the Part 503 Rule has been effective in reducing public exposure to vious framework has been developed for assessing the risks from pathogens relative to 17 yr ago. The percent reduction of both land-applied biosolids, but data have been sparse on the occurrence indicators and pathogens during anaerobic mesophilic digestion of pathogens in biosolids (Eisenberg, 2006). There have been sev- was between 94 and 99% for all organisms, illustrating that eral published reviews on the potential hazards of human patho- such treatment is effective in reducing pathogen loads. gens in biosolids. Of interest is the fact that one review (Straub et al., 1993) was published in 1993 before the promulgation of the USEPA Part 503 Rule (USEPA, 1993) that regulates sewage sludge treatment and land application of the resulting biosolids. A more recent study by Viau and Peccia (2009) quantified genome copies of several pathogens by quantitative polymerase chain reaction Copyright © 2010 by the American Society of Agronomy, Crop Science (qPCR), but this did not provide data on infectious pathogens (i.e., Society of America, and Soil Science Society of America. All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including pho- I.L. Pepper, Univ. of Arizona, Dep. of Soil, Water and Environmental Science, Environmental tocopying, recording, or any information storage and retrieval system, Research Lab., 2601 E. Airport Dr., Tucson, AZ 85756; J.P. Brooks, USDA–ARS, Genetics and without permission in writing from the publisher. Precision Agriculture Unit, P.O. Box 5367, 810 Hwy. 12 East, Mississippi State, MS 39762; R.G. Sinclair, Loma Linda Univ., School of Public Health, Dep. of Environmental Health, J. Environ. Qual. Nichol Hall, Loma Linda, CA 92350; P.L. Gurian, Drexel Univ., Dep. of Civil, Architectural and doi:10.2134/jeq2010.0037 Environmental Engineering, 3141 Chestnut St., 4-270K, Philadelphia, PA 19104; C.P. Gerba, Published online 30 Sept. 2010. Univ. of Arizona, Dep. of Soil, Water and Environmental Science, 1177 E. Fourth St., Shantz Received 28 Jan. 2010. Bldg., Rm. 429, Tucson, AZ 85721. Assigned to Associate Editor Robert Dungan. *Corresponding author ([email protected]). © ASA, CSSA, SSSA 5585 Guilford Rd., Madison, WI 53711 USA Abbreviations: qPCR, quantitative polymerase chain reaction. Table 1. Part 503 Rule pathogen density limits, adapted from USEPA (2000). Historic Data Set Samples of biosolids from Pima County Ina Road Wastewater Pathogen or indicator Standard density limits (dry wt.)† Treatment Plant were collected and analyzed during the period Class A 1988 to 2006. Biosolids resulted from mesophilic anaerobic diges- Salmonella 3 MPN per 4 g tion with a solids content that in the 1980s was approximately Fecal coliforms <1000 MPN per g 2%, compared with the higher solids content of the late 1990s Enteric viruses <1 PFU per 4 g of approximately 6 to 8%. Sampling frequency ranged from Viable helminth ova <1 per 4 g monthly to quarterly. The organisms assayed and the methods of Class B analysis are outlined in Table 2. Of particular interest are the pre- Fecal coliforms <2,000,000 MPN per g 1993 data, which allow for an evaluation of the impact of the Part † MPN, most probable number; PFU, plaque-forming unit. 503 Rule on treatment efficiency. all of the genomes detected could represent viable or nonviable National Data Set organisms). However, qPCR data could be used for a worst-case Between 2005 and 2008 we conducted a national study on risk assessment, since culturable methods will not detect viable the incidence of human pathogens and indicators of patho- but nonculturable organisms. A recent review by Sidhu and Toze gens in anaerobically digested mesophilic biosolids (Class B) (2009) only contained recent data from countries other than the produced within wastewater treatment facilities across the United States, except for one reference to indicator bacteria and United States. Treatment plants generally served metropolitan Salmonella removal (Dahab and Surampalli, 2002). Pathogen areas, most of which had populations >0.5 million and were loadings and removal processes vary in different countries and are located in Arizona (two locations), California (five locations), unlikely to represent those found in the United States (Jimenez Florida (one location), Michigan (one location), Minnesota et al., 2000). Most of the existing studies on occurrence of viable (two locations), Nevada (one location), Oregon (one location), pathogens in biosolids were undertaken before the Part 503 reg- Washington (one location), Wisconsin (three locations), and ulations for treatment of biosolids went into effect (Straub et al., Wyoming (one location). Most samples were “cake” (~20% 1993). Before this time there were no requirements or treatment solids), with some samples being a slurry (~8% solids). All standards, and there was a great deal of variability in treatment samples were immediately shipped overnight on ice to the processes for biosolids at that time. As a result, data collected University of Arizona, where they were analyzed within 24 h. before these regulations went into effect may not reflect current The organisms assayed and the methods of analysis are shown levels of pathogens in biosolids in the United States. This study in Table 3. Many of the pathogens assayed were known to exist presents a large database on the incidence of pathogens and indi- before the Part 503 Rule and data on their incidence before 1993 cators in Class B biosolids, including national and historic distri- are available. Other pathogens, such as Escherichia coli O157:H7, butions. For historic distributions, we monitored pathogens and have emerged after the Part 503 Rule was promulgated. indicators in Class B biosolids from the Ina Road Wastewater Maximum likelihood methods were used to fit lognormal Treatment Plant in Tucson, AZ, on a monthly to quarterly basis distributions to the data. The likelihood function of the data is for 18 yr (Pima County biosolids 1988–2006). For national dis- given by Eq. [1]: tributions, we analyzed current levels of pathogens and indicators N from 21 samplings from 18 wastewater treatment plants from all di 1-di L=P f(log Yi m , s ) F [log(DL)m , s ] [1] major regions of the United States. This is the first nationwide i study of its kind since the USEPA Part 503 Regulations went 2 into effect, and provides a comprehensive database on the cur- where Yi denotes the organism concentration, and m and s rent incidence of pathogens in Class B mesophilic, anaerobically are the national mean and variance. The probability density digested biosolids. The databases presented here not only allow distribution function of a normal distribution is denoted by for a reevaluation of pathogen risks, but also document the vari- f(logYi|m,s) and the cumulative density function of a normal ability of treatment for pathogen removal over time.